My long-term objective is to establish my own laboratory at a research institution to perform breast cancer research with the aim of discovering the missing links between DNA damage repair pathways and breast carcinogenesis which could be exploited for identifying novel therapeutic targets. To accomplish this goal, I joined Dr. Maria Jasin's lab at MSKCC who has performed groundbreaking studies on breast cancer genes including BRCA1, BRCA2 and RAD51 paralogs. Proposed Research: Breast cancer is the most common form of cancer identified among women in the United States. Genetic studies have revealed a direct link between breast/ovarian cancer predisposition and germ-line mutations in BRCA1, BRCA2, PALB2 and paralogs of the recombinase gene RAD51 that are involved in DNA repair by the homologous recombination (HR) pathway. Although the connection between breast cancer and BRCA1 and BRCA2 has been widely investigated, the RAD51 paralogs have been identified only recently as highly penetrant cancer susceptibility genes. Mutants in RAD51 paralog genes in rodent cells are highly sensitive to DNA damaging agents, displays genomic instability and have reduced HR. Biochemically, RAD51 paralogs exists in two different complexes, BCDX2 (RAD51B, RAD51C, RAD51D and XRCC2) and CX3 (RAD51C and XRCC3). However the contributions of individual RAD51 paralogs during early and late stages of HR and the epistatic relationship of BCDX2 and CX3 complexes has not been studied systematically in human cells. The objective of this proposal is to dissect the cellular functions f the RAD51 paralogs in normal human mammary epithelial cells to provide insight into how deficiency of RAD51 paralog genes predisposes individuals to cancer. Specific Aim 1: Identify the functions of the individual RAD51 paralogs in human mammary epithelial cells containing an integrated HR reporter. Specific Aim 2: Determine the epistatic relationship of the RAD51 paralogs to understand the role of the major complexes (BCDX2 and CX3) in HR. Research Design: 1) To identify the functions of RAD51 paralogs in MCF10A DR-GFP cells, I will generate 5 isogenic RAD51 paralog knockouts (RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3) using Transcription Activator-Like Effector Nuclease (TALEN)-directed gene disruptions. 2) To understand the epistatic relationships of BCDX2 and CX3 complexes, I will generate several double-mutant cell lines. I will disrupt members of both complexes (RAD51B-/-XRCC3-/-), disrupt members of only the BCDX2 complex (RAD51B-/- RAD51D-/-), and disrupt members of both complexes using a member common to both complexes (RAD51C-/- ) and one member unique to the BCDX2 complex (RAD51B-/-). The single knockout cell lines and double- mutant cell lines will be assessed for their overall viability, ability to undergo DNA replication, sensitvity to DNA damaging agents, HR proficiency, and genomic instability.